Steroidal compounds, also known as steroids, are a class of compounds with perhydrocyclopentanophenanthrene as mother nucleus and having similar structures. As illustrated below, the basic structure consists of three six-membered rings and a five-membered ring, respectively known as A, B, C, and D rings. Taking the four-membered ring sterane mother nucleus as the matrix, the type, configuration and substitution site of substituents can determine the characters and the functions of different steroidal compounds, and form a series of compounds with unique physiological functions. Among them, the steroid having a hydroxyl substituent at C-3, two methyl groups at C-18 and C-19 and a long hydrocarbon side group mostly in β-configuration at C-17 can be collectively referred to as sterol (3-β sterol), for example, cholesterol, ergosterol and the like. In humans and animals, steroids are the main endogenous hormones, which are secreted by sexual organs and the adrenal cortex, and is closely related to reproduction, brain and bone development, steady state maintenance and regulation of biological effects and so on. As an exogenous hormone, steroid hormone drugs also are a class of indispensable clinical drugs, which play an important role in regulating the body and have extremely important medical value. For example, an adrenocortical hormone has anti-inflammatory, antiallergic, antiallergy, anti-shock response and other effects. In addition, the steroid also has many non-hormonal functions, such as antiviral, neoplastic ailments therapy and the like. Therefore, steroidal drugs are widely used in clinical practice, and are the second largest class of drugs with the production only after antibiotics.

The high demand for steroidal drugs has promoted a vigorous development of another important industry—the extraction and preparation of steroidal drug intermediates (steroidal drugs precursor). The preparation of the steroidal drug intermediate is mainly based on the microbial metabolism of sterol. Mycobacterium has gained widespread attention for its excellent biological metabolism among many microorganisms which could degrade the sterol. Therefore, it's particularly important to analyze the sterol metabolism mechanism of Mycobacterium species. The progress of sterol degradation by Mycobacterium is complicated, and can be divided into two major parts, namely, the degradation of the mother nucleus and the degradation of the side chain. The degradation of the mother nucleus can be divided into the degradation of A and B rings and the degradation of C and D rings, wherein, the research on the degradation mechanism of A and B rings is more adequate and detailed than that of C and D rings.
The acyl-CoA dehydrogenase, as shown in FIG. 1, was speculated by the researchers to be one of the key enzymes involved in degradation of the C and D rings of sterol nucleus, which can participate in the dehydrogenation reaction during the degradation of the C and D rings. The C and D rings are opened and eventually degraded to form CO2 and H2O. Therefore, deletion of the gene encoding acyl-CoA dehydrogenase in Mycobacteria is likely to prevent the degradation process of the C and D rings, thereby producing valuable steroidal drugs intermediates. Until now, valuable intermediates produced from sterol metabolized by Mycobacteria can be mainly divided into C19-steroids (AD, ADD, 9-OHAD, testosterone, boldenone) and C22-steroids (20-carboxy-pregn-4-en-3-one, 4-BNC; 20-hydroxymethyl-pregn-4-en-3-one, 4-BNA; and corresponding 9α-hydroxylation and/or C1 (2) dehydrogenation compounds of each substance and so on). Since the degradation mechanisms of C and D rings have not been thoroughly analyzed, the Mycobacteria strains used to produce the steroidal drug intermediates that retain only the C and D rings are very rare. A valuable compound that can be used for commercialization in the degradation of C and D rings is a lactonization product (sitolactone, HIL) of 5-OHHIP which retains the complete C and D, and it can be used in production of mifepristone, estrogen, and the other steroid drugs. In the steroid medical industry, for the production of sitolactone, the Nocardia bacteria are used to ferment sterol to form 5-OHHIP, followed by pH adjustment, so that 5-OHHIP is lactonized to form commercial sitolactone. The sitolactone production strain is relatively inefficient, and the steps are cumbersome, and thus it has certain defects.
Therefore, it's necessary to study the acyl-CoA dehydrogenase gene to overcome the problems that the degradation mechanism of C and D rings that are unknown, and the sitolactone production strain is lacking, and thus to achieve the transformation and development of high-efficiency production strain, enrich the types of valuable intermediates, improve the production efficiency and product quality of steroids drugs, reduce energy consumption in the steroid drugs production, simplify production steps, and reduce production costs.